MX2013009608A - Cystamine analogues for the treatment of parkinson's disease. - Google Patents

Abstract

The present invention relates to the use of cystamine analogues for the treatment of Parkinson's disease. The present invention also relates to the use of composition comprising cystamine analogues and cysteine. The present invention relates to a method for modifying the progression of Parkinson's disease comprising administering a therapeutically effective amount of at least one cystamine analogue or a pharmaceutically acceptable salt of a cystamine analogue to a patient in need thereof.

Description

ANALOGUES OF CYSTAMINE FOR THE TREATMENT OF THE DISEASE OF PARKINSON Background of the Invention Current treatments for Parkinson's disease (PD) are symptomatic for the most part and do not prevent the neuronal degeneration that is causing the progress of the disease. The properties of cystamine in Parkinson's disease and in Huntington's disease have been studied in several animal models. In animal models of Huntington's disease (HD), cystamine has shown neuroprotective effects by prolonging the life span and decreasing the motor symptoms of mice carrying the gene for Huntington's disease (Dedeoglu et al. 2002; Karpuj et al. 2002). In vitro and in vivo evidence has shown the ability of cystamine to inhibit transglutaminase, an enzyme involved in protein aggregates such as the mutated form of the huntingtin protein (Green 1993, Jeitner et al 2005, Wang et al 2005). The increase in brain levels of brain-derived neurotrophic factor (BDNF) has also been accurately determined as one of the key elements of this neuronal protective effect (Borrell-Pages et al. 2006). A Ref. 243106 High dose of cystamine supplied through drinking water attenuates the oxidative stress and the detrimental effect of 1-methyl-4-phenyl-1,2,3, β-tetrahydropyridine (MPTP) on mitochondrial functions (Stack et al. 2008) . The effects of cystamine and / or cysteamine have been reported in the MPTP mouse model of Parkinson's disease (Sun et al 2010, Tremblay et al 2006, Stack et al 2008, Gibrat et al 2010).

The metabolism of cystamine generates several intermediate products that include not only cysteamine, but also hypotaurine and taurine. Cystamine and cysteamine are both organic compounds and were initially described as radioprotectants (Bacq and Beaumariage 1965). Although cysteamine is the decarboxylated form of cysteine, the main source results from its constitutive production by all tissues via the degradation of coenzyme A (Pitari et al. 1992), which is involved in metabolic processes notably in the generation of ATP through the Krebs cycle (Leonardi et al. 2005). Although cysteine is a common constituent of most proteins (Lee et al. 2004), plasma levels of basal cysteine are usually low because their thiol is susceptible to oxidation and leads to the disulfide cystine derivative.

Cysteamine, the reduced form of cystamine (2-aminoet-anotiol) is approved for the treatment of cystinosis, a childhood disorder which causes renal failure through the intracellular accumulation of cystine (Dohil et al. 2009). Because cysteamine has shown significant efficacy in mouse models of. Huntington's disease (Borrell-Pages et al. 2006) and its safety has been documented, the molecule is currently in development for patients suffering from this disorder (Dubinsky and Gray 2006).

A preliminary trial with cysteamine bitartrate (CYSTAGONMR) was recently performed in patients with Huntington's disease, in part to establish a safe therapeutic dose (Dubinsky and Gray, 2006). Nine patients with Huntington's disease enrolled in this phase I clinical trial with known, single-center identity. The subjects received a treatment with cysteamine of 10 mg / kg per day with a weekly increase of 10 mg / kg per day up to a maximum dose of 70 mg / kg or until the development of intolerable side effects (nausea and motor deterioration). The trial concluded that a dose of 20 mg / kg per day of cysteamine was tolerable in people suffering from Huntington's disease (Dubinsky and Gray, 2006). However, clinical efficacy was not demonstrated. Even if they are not fully transferable to humans, the Studies carried out in animal models of Huntington's disease showed that much higher doses of cystamine or cysteamine were required to achieve a significant therapeutic effect. Additionally, although cysteamine can cross the blood-brain barrier (BBB), it takes larger doses of cystamine or cysteamine (ip or po) to detect a variation in cysteamine or its metabolites in the brain (Bousquet et al. , 2010). The effectiveness of cystamine and cysteamine in modifying the progress of Parkinson's disease, as well as its transport properties in the brain are unknown.

The existing therapies for Parkinson's disease are designed mainly for the management of symptoms and until now there is no available treatment to attenuate the progress of the disease. Therefore, there is a need for the development of therapeutic agents that can modify the rate of progression of Parkinson's disease.

The inventors have demonstrated, for the first time, that cystamine analogs can be used to modify the progress of Parkinson's disease.

Brief Description of the Invention The present invention provides a method for modifying the progress of Parkinson's disease, the The method comprises administering a therapeutically effective amount of at least one cystamine analog, a pharmaceutically acceptable salt thereof, a composition or a combination of the invention to a patient in need thereof.

The present invention provides the use of a therapeutically effective amount of at least one cystamine analog, a pharmaceutically acceptable salt thereof, a composition or a combination of the invention to modify the progress of Parkinson's disease in a patient in need thereof. .

The present invention provides the use of a therapeutically effective amount of at least one cystamine analogue or a pharmaceutically acceptable salt thereof as a neurorescue agent and / or as a neurorestoration agent to modify the progress of Parkinson's disease in a patient.

The present invention provides a combination or a pharmaceutical composition for modifying the progress of Parkinson's disease comprising at least one cystamine analogue or a pharmaceutically acceptable salt thereof and comprising cysteine or a pharmaceutically acceptable salt thereof.

The present invention provides a combination or a pharmaceutical composition for modifying the progress of the Parkinson's disease comprising at least one cystamine analogue or a pharmaceutically acceptable salt thereof as a neurorescue agent and / or a neurorestoration agent and comprising cysteine or a pharmaceutically acceptable salt thereof.

In yet another aspect, there is provided the use of a cystamine analog, or a pharmaceutically acceptable salt thereof, a composition or a combination of the invention for the manufacture of a medicament as described herein.

Brief Description of the Figures Figures 1A-1C: Beneficial effects of cystamine on nigral neurons positive for tyrosine hydroxylase.

Figure 1A. Stereological counts of neurons cells positive for tyrosine hydroxylase (TH) in the substantia nigra pars compacta (SNpc) revealed a significant decrease in the total number of TH-positive neurons in PTP mice treated with saline, compared to animals treated with saline solution + saline solution (p <0.001). Figure 1A. Mice treated with Cystamine Pre and Post-MPTP demonstrated a similar number of TH-positive neurons than animals treated with saline (titrated: "TH-positive null neurons"). Figure IB. Photomicrographs of the SNpc that show a high number (comparable to the saline solution) of neurons stained with Crosyl and positive for TH in saline and mice treated with post-MPTP cystamine compared to mice treated with MPTP saline. The table in Figure 1C recapitulates the stereological counts of Cresil and TH cells. The lower panels illustrate the timelines of the pre- and post-treatment schedules. The values are expressed as means ± S.E.M. Statistical analyzes were performed using a unidirectional ANOVA analysis. Significant difference with the group treated with saline + saline: *** = p < 0.001. Significant difference with the group treated with MPTP + saline: # = p < 0.05; ## = p < 0.01; ### = p < 0.001. Scale bar in Figure IB = 400 μp ?, Insertion = 25 μp ?. Abbreviations: Pre-Tx (treatment with pre-MPTP cystamine); Pos-Tx (treatment with post-MPTP cystamine).

Figures 2A-2B: Beneficial effect of cystamine on the expression of Nurrl nigral mRNA.

Figure 2A. Densitometric measurements of Nurrl mRNA levels (a gene involved in the expression and maintenance of the dopamine (DA) phenotype) in the SNpc revealed that the levels of 3 control groups (saline + saline, saline + cystamine Pre- Tx, saline + cystamine Pos-Tx) and MPTP animals treated with cystamine were similar, while the levels of Nurrl mRNA in MPTP animals treated with saline were significantly decreased (p <0.01) (the title of which is: "Nurrl mRNA levels"). Figure 2B. Photomicrographs at the SNpc level (see arrow) illustrate normal levels of Nurrl mRNA in control and post-MPTP treated cistamine mice compared to mice treated with MPTP saline solution Figure 2B. The values are expressed as means ± S.E.M. Statistical analyzes were performed using a unidirectional ANOVA analysis. Significant difference with the group treated with saline + saline: ** = p < 0.01. Significant difference with the group treated with MPTP + saline: # = p < 0.05; ## = p < 0.01. Scale bar in Figure 2B = 1 mm.

Figures 3A-3C: Beneficial effect of cystamine on nigral cells positive for DAT.

The expression of the DA transporter mRNA (DAT) was also revealed by. means of in situ hybridization. Figure 3A. Stereological cell counts of cells expressing DAT in the SNpc showed a significant decrease in the total number of neurons in MPTP mice treated with saline, compared to animals treated with saline + saline (p <0.001). Figure 3A. Mice treated with cystamine pre and post-MPTP showed a comparable number of cells positive for DAT as the animals treated with saline solution (whose title is: "Positive nigral neurons for DAT"). Figure 3B. Photomicrographs of the SNpc represent cells that express DAT mRNA. The insert represents the autoradiography of DAT mRNA before the emulsion (measured by means of densitometry). The table in Figure 3C recapitulates the stereological counts of cells and densitometric measurements of the expression of DAT mRNA in the SNpc. The values are expressed as means ± S.E.M. Statistical analyzes were performed using the unidirectional ANOVA analysis. Significant difference with the group treated with saline + saline: * = p < 0.05; *** = p < 0.001. Significant difference with the group treated with MPTP + saline: # = p < 0.05; ## = p < 0.01. Scale bar in Figure 3B = 400 μta, insert = 500 um.

Figures 4A-4C: Temporal course of the loss of positive cells for nigral TH in the subacute MPTP model.

Figure 4A. Stereological cell counts of HT-positive neurons in the SNpc revealed a significant decrease in the total number of neurons positive for TH 7 and 14 days after the last MPTP injection compared to the group treated with saline (p <0.01). ) and the post-MPTP group 1 day, which only showed a trend towards a decreased number of neurons (p = 0.063) (whose title is: "Nigral neurons positive for TH"). Figure 4B. Photomicrographs of the SNpc represent a reduced number of neurons stained with Cresil and positive for TH in the post-MPTP groups 7 and 14 days. The table in Figure 4C recapitulates the stereological counts of Cresil and TH cells. The values are expressed as means + S.E.M. Statistical analyzes were performed using a unidirectional ANOVA analysis. Significant difference with the group treated with saline: ** = p < 0.01. Scale bar in Figure 4B = 400 μ ??.

Figures 5A-5B: Time course of decreases in the expression of Nurrl and DAT mRNA in the subacute MPTP model.

The densitometric measurements of the expression of MRNA Figure 5A Nurrl and Figure 5B DAT showed significantly decreased levels of both markers of DA in the SNpc starting 24 hours after treatment with MPTP (p <0.01 and p <0.05 respectively). The values are expressed as means ± S.E.M. Statistical analyzes were performed using a unidirectional ANOVA analysis. Significant difference with the control group: ** = p < 0.01. * = P < 0.05 (whose titles are: Figure 5A "Expression of Nurrl Nigral mRNA" and Figure 5B "Expression of Dig Nigral mRNA").

Figures 6A-6C: Temporal course of the apoptotic process of nigral DA in the subacute MPTP model.

Western Blot analysis of protein levels Figure 6A BAX and Figure 6B Bcl-2 in the ventral mesencephalon. Figure 6C. The BAX / Bcl2 ratio increases significantly 24 hours after the last MPTP injection (p <0.05) suggesting that, with this specific MPTP delivery regimen, an apoptotic process has already begun at this time. The values are expressed as means ± S.E.M. The statistical analyzes were performed using the unidirectional ANOVA analysis. Significant difference with the control group: * = p < 0.05 (whose titles are: Figure 6A "Levels of BAX proteins in the ventral mesencephalon", Figure 6B "Levels of Bcl2 proteins in the ventral mesencephalon" and Figure 6C "Ratio of BAX / Bcl2").

Figures 7A-7C: Increased levels of cysteamine in the brain. Brain levels of cysteamine Figure 7B and cysteine Figure 7C measured by means of HPLC coupled with fluorescence detection. The molecular structures and HPLC elution profiles of a standard solution of cysteamine (2) and cysteine (1) are shown in Figure 7A. Cysteamine is significantly increased in response to an i.p. individual dose of 50 mg / kg in mice sacrificed 1 hour after injection, compared to vehicle-treated mice killed in the same time point (p <0.05) Figure 7B. The dose of 200 mg / kg also causes a significant increase in cysteamine 1 hour and 3 hours after injection of cystamine (p <0.01) Figure 7b. The cerebral levels of cysteine are stable regardless of the doses and infusion times Figure 7C. The data are expressed as means (nmol / mg of protein) ± S.E. . * p < 0.05; ** p < 0.01.

Figures 8A-8C: Constant levels of hipotaurine and taurine in the brain. Brain Concentrations of Hypoturin Figure 8B and Taurine Figure 8C measured by HPLC coupled with UV detection. The molecular structures and elution profiles of HPLC of a standard solution containing 1 ng / mL of taurine (1) and hypotaurine (2) are shown in Figure 8A. Stable measurements of hipotaurine Figure 8B and taurine Figure 8C were observed in the brain. The data are expressed as means (nmol / mg of protein) ± S.E.M.

Figures 9A-9C: Increased uptake in the brain of cysteamine in the presence of cysteine. Demonstration of cysteine and cysteine brain uptake using a cerebral perfusion technique in situ and quantification by means of an HPLC method. Schematic illustration of the cerebral perfusion method in situ Figure 9 ?. A catheter is inserted directly into the carotid artery, internal, right to ensure that 100% of the perfusate reaches the right hemisphere after the appropriate ligatures (blue vessels) Figure 9A. Both the cysteine Figure 9B and the cysteamine Figure 9C can cross the BBB as demonstrated by the high clarification coefficient of each molecule (μ?, / Q / s). When co-perfused, the clearance coefficients of cysteine and cysteamine increase significantly. The data are expressed as means + S.E.M. (μ?, / q / s) * p < 0.05 (whose titles are: Figure 9B "Cysteine uptake in the brain" and Figure 9C "Cysteamine uptake into the brain").

Figures 10A-10E: Cystamine rescues dopaminergic neurons in the mouse model of unilateral striatal ß-OHDA from Parkinson's disease. The panel in Figure 10E illustrates the time course of the experiment. All mice were subjected to a unilateral intrastriatal injection of 6-OHDA (4 ug) or an equivalent volume of vehicle (sham). Three days later, during the ongoing dopaminergic degenerative process, the mice received a 10 mg / kg treatment of cystamine (or saline) daily for 14 days. A transcardiac perfusion was performed on the mice 24 hours after the last injection of cystamine and the brains were processed for post mortem analysis. Figure 10A. A stereological cell count of neurons positive for TH in the SNpc revealed a significant decrease of 72% in the total number of TH-positive neurons in 6-OHDA mice treated with saline, compared to animals treated with sham + saline (p <0.001). The 6-OHDA mice treated with cystamine exhibited only a 27% decrease in the total number of HT-positive neurons compared to animals treated with sham + cystamine (p <0.05). The group treated with 6-OHDA + saline was significantly different from the group treated with 6-OHDA + cystamine (p < 0.001). Figure 10B and Figure 10C. Densitometric measurements for the levels of Nurrl and DAT mRNA in the SNpc, respectively. These 2 additional markers of dopaminergic integrity revealed the same pattern observed for TH staining in Figure 10A, confirming the neurorescue properties of cystamine. Figure 10D. Measurements of striatal DA content performed by HPLC showed significantly decreased DA levels only in 6-OHDA mice treated with saline compared to the control mice (p <0.01). The values are expressed as means ± S.E.M. Statistical analyzes were performed using a unidirectional ANOVA analysis. * = p < 0.05; ** = p < 0.01; *** = p < 0.001 (whose titles are: Figure 10A "Nigral neurons positive for TH", Figure 10B "Expression of Nurrl Nigral mRNA", Figure 10C "Expression of DAT Nigral mRNA", Figure 10D "Stratial doparain levels").

Figures 11A-11D: Cystamine reverses behavioral impairments induced by striatal 6-OHDA lesions in mice. The panel of Figure 11D illustrates the time course of the experiment. All mice were subjected to a unilateral intrastriatal injection of 6-OHDA (4 ug) or an equivalent volume of vehicle (sham). Three weeks later, when the lesion was stable and had reached maximum degeneration, the mice received a treatment of 10 mg / kg of cystamine (or saline) daily for 6 weeks. Throughout the experiment, the mice were evaluated with 3 different behavioral tests at 3 different time points: before the start of treatment with cystamine (3 weeks after surgery), 6 weeks and 9 weeks after the injury. Behavioral tests were selected to evaluate sensorimotor asymmetries that are indicative of the degree of unilateral dopaminergic lesion. Figure 11A. Contralateral rotations induced by cumulative apomorphine were measured with a rotameter apparatus. The unilateral 6-OHDA lesion induced an increase in contralateral rotations 3, 6 and 9 weeks after lesion significantly attenuated by treatment with cystamine 6 and 9 weeks, after surgery (p <0.05). Figure 11B. The displacement test revealed a decrease in the Percentage of adjustment of steps for the contralateral anterior leg (compared to homolateral). Figure 11C. The asymmetry of limb use was also shown for the cylinder test in which mice injured by 6-OHDA showed a significant decrease in the percentage of contralateral contacts at 3 weeks, 6 weeks and 9 weeks after surgery compared to the mock mice. This asymmetry was not visible for mice injured by 6-OHDA treated with cystamine at 6 and 9 weeks after injury. The values are expressed as means + S.E.M. Statistical analyzes were performed using a unilateral ANOVA analysis. * = P < 0.05; ** = p < 0.01; *** | = p < 0.001 (whose titles are: "Drug-induced motor behavior" Figure 11A "Apomorphine-induced rotations", "Spontaneous motor behaviors" Figure 11B "Step adjustment test (displacement)", Figure 11C "Limb use asymmetry test (cylinder)").

Figures 12A-12D: Cystamine restores some aspects of the dopaminergic nigral system and changes the striatal catecholaminergic contents and dynamics in the unilateral striatal 6-OHDA mouse model of Parkinson's disease. The panel of Figure 12D illustrates the time course of the experiment. As shown in Figure 11, all mice were subjected to an intrastriatal, unilateral injection of 6-OHDA (4 ug) or a volume Vehicle equivalent (sham) and treatment with cystamine (or saline) started 3 weeks later for a period of 6 weeks. One was made. transcardiac perfusion to the mice 24 hours after the last injection of cystamine and the brains were processed for post mortem analysis. Figure 12A. The stereological cell count of TH-positive neurons in the SNpc revealed a significant decrease of 93% in the total number of TH-positive neurons in 6-OHDA mice treated with saline, compared to mock animals + saline (p. < 0.001). The 6-OHDA mice treated with cystamine exhibited only a 65% decrease in the total number of HT-positive neurons compared to sham + cystamine animals (p <0.001). The 6-OHDA + saline group is significantly different from the 6-OHDA + cystamine group (p < 0.01). Figure 12B. The DA levels of cystamine measured by HPLC in mice injured by 6-OHDA were decreased by 88% and 84% for the animals treated with saline and cystamine respectively (p <0.001). Cystamine did not significantly restore the DA content in the injured striatum. Figure 12C. However, cystamine exerted a strong tendency to normalize DA production compared to 6-OHDA mice treated with saline, which correlates significantly with the behavioral injury evaluation tests (data not shown). The values are expressed as means ± S.E.M. Statistical analyzes were performed using a unidirectional ANOVA analysis. * = P < 0.05; ** = p < 0.01; *** = p < 0.001 (whose titles are: Figure 12A "Nigral neurons positive for TH", Figure 12B "Striatal dopamine levels" and Figure 12C "Striatal dopamine production").

Detailed description of the invention Uses and Methods In one embodiment, the cystamine analogs and pharmaceutically acceptable salts thereof can be used as neurorecovery and / or neuroreconstitution agents. This activity of neurorecovery / neuroreconstruction can be distinguished from the activity of a neuroprotective agent.

As used in this document, "a neuroprotective agent" can protect the remaining "healthy" neurons from the degenerative process. Therefore, it can be seen that a neuroprotective agent could be administered at the time of diagnosis.

As used in this document, "a neurorescue agent" can stop the neurodegenerative process in neurons that are injured, but not dead, with or without functional recovery. Therefore, you can understand that a neurorescue agent should be administered as soon as possible, but can be administered after PD diagnosis.

As used herein, "a neuroreconstitution agent" can restore a function by means of a functional and / or structural restoration and regeneration of injured neurons. Therefore, it can be seen that a neurorecovery agent is highly relevant for clinical use in PD since it can show maximum efficacy after diagnosis.

In a further embodiment, the cystamine analogs and pharmaceutically acceptable salts thereof can be used to modify the progress of Parkinson's disease.

In one embodiment, "modifying the progress of Parkinson's disease" is characterized by a) a reduction of the neurodegenerative process by an anti-apoptotic action on neurons that are injured, but not dead, with or without a functional recovery; and / or b) functional and / or structural restoration and regeneration.

In an additional modality, "modifying the progress of Parkinson's disease" is characterized by one of the following mechanisms: a) a reduction of the neurodegenerative process by an anti-apoptotic action on neurons that are injured, but not killed, with or without a functional recovery; I b) a functional and / or structural restoration and regeneration of injured neurons, and / or c) the promotion of neurogenesis.

In yet another modality, the progress of Parkinson's disease is quantified by means of the classification of the Unified Parkinson's Disease Classification Scale (Total UPDRS), an increase in the score of Total UPDRS represents a progression of the symptoms of Parkinson's disease and the increase in the rating of Total UPDRS over a period of time represents the rate of progression of Parkinson's disease. See for example: Goetz CG, Tilley BC, Shaftman SR, Stebbins GT, Fahn S, Martinez-Martin P, Poewe W, Sampaio C, Stern MB, Dodel R and collaborators (2008) Movement Disorder Society-sponsored review of the Unified Parkinson 's Disease Rating Scale (MDS-UPDRS): scale presentation and clinimetric testing results. Mov Disord 23: 2129-2170.

In yet another embodiment of this method, the time period is 12, 24 or 36 weeks after the start of administration of a cystamine analogue or a pharmaceutically acceptable salt thereof.

As used in this document, the stages of a Patients with Parkinson's disease are described by Hoehn and Yahr in the following five different stages depending on the symptoms (Hoehn M M, Yahr D, Parkinsonism: onset, progression and mortality, Neurology 1967, 17: 427-42).

Stage I: (mild or initial illness): Symptoms affect only one side of the body.

Stage II: Both sides of the body are affected, but the posture remains normal.

Stage III: (moderate illness): Both sides of the body are affected and there is a slight imbalance during standing or displacement. However, the person remains independent.

Stage IV: (advanced disease): Both sides of the body are affected and there is a disabling instability while standing or moving. The person in this stage requires substantial help.

Stage V: A serious, fully developed disease is present. The person is restricted to a bed or a chair.

As used herein, a "patient with early-stage Parkinson's disease" is a patient with Parkinson's disease in Stage I or II of Parkinson's disease as defined by Hoehn and Yahr, and who does not require symptomatic anti-disease therapy Parkinson. In one modality, this patient with Parkinson's disease does not require symptomatic treatment for at least the next 9 months. A patient with early-stage Parkinson's disease can be identified as such by performing a relevant test.

In one modality, the patient is a patient with Parkinson's disease in the initial stage.

In yet another modality, the patient with Parkinson's disease in the initial stage is a Stage I patient according to the classification of Hoehn and Yahr.

In yet another modality, the patient with Parkinson's disease in the initial stage is a patient who has a total score of UPDRS less than 30; less than 25; less than 23; less than 21; or less than 20.

In still another modality, the patient with Parkinson's disease is a patient in Stage I, II, III, IV or V according to the classification of Hoehn and Yahr.

In still another modality, the patient with Parkinson's disease is a patient in Stage III, IV or V according to the classification of Hoehn and Yahr.

In yet another modality, the patient with Parkinson's disease is a Stage III or IV patient according to the classification of Hoehn and Yahr.

In yet another modality, the patient with Parkinson's disease is in Stage III according to the classification of Hoehn and Yahr.

The present invention relates to uses or methods for: reduce fatigue in a patient with Parkinson's disease; reduce the severity of non-motor symptoms in a patient with Parkinson's disease; reduce functional decline in a patient with Parkinson's disease; reduce the clinical progress of the disease; or slow down the clinical progress of the disease.

The present invention still further provides a method for treating a patient exhibiting Parkinson's disease, comprising the identification of patients exhibiting Parkinson's disease and periodically administering to the patient thus identified an amount of a cystamine analogue or a pharmaceutically acceptable salt thereof or a composition or a combination of the invention that is effective to treat the patient.

The present invention relates to a method for slowing down or reducing the progress of Parkinson's disease in a patient comprising administering to the patient a therapeutically effective amount of minus a cystamine analog or a pharmaceutically acceptable salt thereof, a composition or a combination of the invention.

The present invention relates to the use of a therapeutically effective amount of at least one cystamine analog or a pharmaceutically acceptable salt thereof, a composition or a combination of the invention to slow down or reduce the progress of Parkinson's disease in a patient.

The present invention further provides the use of a therapeutically effective amount of a cystamine analogue or a pharmaceutically acceptable salt thereof or a composition or combination of the invention to slow the clinical progress of Parkinson's disease in a patient with the Parkinson's disease.

The present invention further relates to a method for slowing the rate of clinical progress of Parkinson's disease in a patient with Parkinson's disease comprising administering to the patient a therapeutically effective amount of a cystamine analogue or a pharmaceutically acceptable salt thereof. or a composition or a combination of the invention.

The present invention still further provides for the use of a therapeutically effective amount of a cystamine analog or a pharmaceutically acceptable salt or a composition or a combination of the invention to reduce the severity of non-motor symptoms in a patient with Parkinson's disease.

The present invention still further provides a method for reducing the severity of non-motor symptoms in a patient with. Parkinson's disease which comprises administering to the patient a therapeutically effective amount of a cystamine analogue or a pharmaceutically acceptable salt or a composition or a combination of the invention.

The present invention still further provides a method for treating a patient having Stage I, II, III, IV or V of Parkinson's disease (according to the Hoehn and Yahr classification), comprising the identification of patients who exhibit Parkinson's disease and periodically administer to the patient thus identified an amount of a cystamine analog or a pharmaceutically acceptable salt thereof, or a composition or combination of the invention that is effective in treating the patient.

The present invention further provides a cystamine analog or a pharmaceutically acceptable salt thereof, or a composition or combination of the invention for use in the treatment of a patient exhibiting early symptoms of Parkinson's disease.

The present invention still further provides a pharmaceutical composition comprising a pharmaceutically effective amount of a cystamine analog or a pharmaceutically acceptable salt thereof, or a composition or combination of the invention for use in reducing the rate of progress of the Parkinson's disease in a patient with early-stage Parkinson's disease.

The present invention still further provides the use of a therapeutically effective amount of a cystamine analogue or a pharmaceutically acceptable salt thereof or a composition or combination of the invention for treating a patient exhibiting early symptoms of Parkinson's disease.

The present invention still further provides a method for treating a patient exhibiting early symptoms of Parkinson's disease, comprising the identification of patients exhibiting early symptoms of Parkinson's disease and periodically administering to the patient thus identified an amount of a cystamine analog or a pharmaceutically acceptable salt thereof, or a composition or combination of the invention that is effective in treating the patient.

The present invention further provides a cystamine analogue or a pharmaceutically acceptable salt thereof or a composition or a combination of the invention for the 7 Use in the reduction of fatigue in a patient with Parkinson's disease in early stage.

The present invention still further provides for the use of a therapeutically effective amount of a cystamine-analog or a pharmaceutically acceptable salt thereof or a composition or combination of the invention to reduce fatigue in a patient with early-stage Parkinson's disease. .

The present invention still further provides a method for reducing fatigue in a patient with early-stage Parkinson's disease, which comprises identifying a patient who is a patient with early stage Parkinson's disease and periodically administering the identified patient of this way an amount of a cystamine analog or a pharmaceutically acceptable salt thereof is effective to reduce fatigue.

The present invention still further provides a method for reducing the severity of non-motor symptoms in a patient with early stage Parkinson's disease, which comprises identifying a patient who is a patient with early-stage Parkinson's disease and periodically administering it to the patient. patient identified in this way an amount of a cystamine analog or a pharmaceutically acceptable salt thereof or a composition or a combination of the invention that is effective in reducing the severity of non-motor symptoms.

The present invention further provides a method for reducing fatigue in a patient with early-stage Parkinson's disease, which comprises periodically administering to a patient with early-stage Parkinson's disease an amount of a cystamine analog or a pharmaceutically acceptable salt. of the same or a composition or a combination of the invention that is effective in reducing fatigue.

The present invention further provides a method for reducing the severity of non-motor symptoms in a patient with early-stage Parkinson's disease, comprising periodically administering to a patient with Parkinson's disease at the initial stage an amount of a cystamine analog or a pharmaceutically acceptable salt thereof or a composition or combination of the invention which is effective in reducing the severity of non-motor symptoms.

The present invention further provides a method for slowing the clinical progress of Parkinson's disease in a patient with Parkinson's disease comprising periodically administering to the patient with Parkinson's disease an amount of a cystamine analogue or a pharmaceutically acceptable salt. thereof, or a composition or a combination of the invention which is effective in decreasing the rate of clinical progress of Parkinson's disease in the patient.

The present invention further provides a cystamine analog or a pharmaceutically acceptable salt thereof, or a composition or a combination of the invention for use in reducing the rate of progression of Parkinson's disease in a patient with Parkinson's disease. in the initial stage.

The present invention further provides a cystamine analogue or a pharmaceutically acceptable salt thereof, or a composition or a combination of the invention for use in reducing functional decay in a patient with early-stage Parkinson's disease.

Pharmaceutical Compositions and Analogs of Cistamine In one aspect, the cystamine analogue is cysteamine, cystamine, taurine or hypothaurin or a pharmaceutically acceptable salt thereof.

In a further aspect, the cystamine analog is cystamine or cysteamine or a pharmaceutically acceptable salt thereof.

In a further aspect, the cystamine analogue is cysteamine bitartrate.

In a further aspect, the cystamine analogue is cysteamine hydrochloride.

In a further aspect, the cystamine analogue is cystamine hydrochloride.

In another aspect, there is provided a pharmaceutical composition comprising at least one cystamine analog or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier or excipient.

In another aspect, a combination comprising a cystamine analog or a pharmaceutically acceptable salt thereof and one or more additional agents such as bromocriptine, benzotropin, levodopa, ropinirole, pramipexole, rotigotine, cabergoline, entacopona, tolcapone, amantidine, selegiline is provided. and rasagiline.

In yet another aspect, there is provided the use of a cystamine analog, or a pharmaceutically acceptable salt thereof, a composition or a combination of the invention for the manufacture of a medicament for modifying the progress of Parkinson's disease in a patient.

According to a further embodiment, the compounds of the present invention are represented by the following formulas: NH2- (CH2) 2-SH Cysteamine NH2- (CH2) 2-S-S- (CH2) 2-NH2 Cistamine NH2- (CH2) 2-S (02) -OH Taurine NH2- (CH2) 2-S (0) -0H Hipotaurine L-cysteine or pharmaceutically acceptable salts thereof.

Cystamine analogs or a pharmaceutically acceptable salt thereof can be obtained by methods well known in the art. The compounds are available from different sources, for example, from Sigma-Aldrich, St. Louis, O. EUA.

In one embodiment, the present invention provides a pharmaceutical composition comprising at least one cystamine analog or a pharmaceutically acceptable salt thereof described herein and further comprising at least one additional agent wherein the additional agent is cysteine .

In one embodiment, the present invention provides a pharmaceutical composition comprising at least one cystamine analogue or a pharmaceutically acceptable salt thereof described herein and further comprising at least one additional agent wherein the additional agent is L-cysteine.

In another embodiment, a combination comprising at least one cystamine analog described herein and one or more additional agents is provided.

In one embodiment, where the additional agent is cysteine.

In a modality, where the additional agent is L-cysteine.

In one embodiment, the cystamine and cysteine analog are present in a 10: 1 to 1:10 ratio of cystamine and cysteine analog, respectively. In a further embodiment, the cystamine and cysteine analog are present in a ratio of 1: 1.

In a combination modality, the cystamine analog and the additional agent are administered or are suitable for sequential use.

In another combination mode, the cystamine analog and the additional agent are administered or are suitable for simultaneous use.

The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation and thus the formulations Pharmaceuticals comprising a combination as defined above together with a pharmaceutically acceptable carrier therefore comprise a further aspect of the invention.

The individual components for use in the method of the present invention or the combinations of the present invention can be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.

In one embodiment, the present invention provides the use of a compound, a composition or a combination as described herein for the manufacture of a medicament.

Unless stated otherwise, the structures depicted herein are also made to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, the double bond isomers (Z) and (E) and the conformational isomers (Z) and (E). Therefore, the individual stereochemical isomers as well as the enantiomeric, diastereomeric and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. The optical isomer Individual or enantiomer can be obtained by a method well known in the art, such as chiral HPLC, enzymatic resolution and a chiral auxiliary.

, In one embodiment, where applicable, the cystamine analogues or the cistern are provided in the form of an individual stereoisomer at least 75%, 85%, 90%, 95%, 97% and 99% free of the corresponding stereoisomers .

Pharmaceutically acceptable salts of the cysteine or cysteine analogs are also provided. By the term pharmaceutically acceptable salts of the compounds is meant those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, trifluoroacetic, citric, methanesulfonic, formic, benzoic acid , malonic, naphthalene-2-sulphonic and benzenesulfonic. Other acids such as oxalic acid, while by themselves are not pharmaceutically acceptable, may be useful as intermediates in the preparation of cystamine analogs and their pharmaceutically acceptable acid addition salts.

Salts derived from amino acids are also included (for example, L-arginine, L-lysine).

Salts derived from appropriate bases include alkali metals (e.g., sodium, lithium, potassium) and alkaline earth metals (e.g. calcium, magnesium).

A reference hereinafter to the cysteine or cysteine analogs includes that compound and its pharmaceutically acceptable salts.

In one embodiment, the salt is a bitartrate salt.

In one embodiment, the salt is a hydrochloride salt.

With respect to pharmaceutically acceptable salts, see also the list of commercially promoted salts approved by the FDA found in Table I of Berge et al., Pharmaceutical Salts, J. of Phar. Sci., Volume 66, no. 1, January 1977, pages 1-19, the description of which is incorporated herein by way of reference.

Those skilled in the art will appreciate that the compounds can exist in different polymorphic forms. As is known in the field, polymorphism is a capacity of a compound to crystallize as more of a distinct crystalline or "polymorphic" species. A polymorph is a solid crystalline phase of a compound with at least two different arrays or polymorphic forms of that molecule of compound in the solid state. The polymorphic forms of any given compound are defined by the same chemical formula or composition and are as different in the chemical structure as the crystalline structures of two different chemical compounds.

Those skilled in the art will appreciate that the compounds according to the present invention can exist in different forms of solvate, for example hydrates. Solvates of cystamine analogues or cysteine can also be formed when the solvent molecules are incorporated into the crystalline lattice structure of the compound molecule during the crystallization process.

Unless defined otherwise, all technical and scientific terms used in this document have the same meaning as commonly understood by a person of ordinary experience in the field to which this invention pertains. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

For the purposes of this invention, the chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, the general principles of organic chemistry are described in "Organic Chemistry" , Thomas Sorrell, University Science Books, Sausalito: 1999, and '"March' s Advanced Organic Chemistry", 5th Ed., Ed .: Smith, MB and March, J., John Wiley & Sons, New York: 2001.

Additionally, unless stated otherwise, the cysteine or cysteine analogs depicted herein are also made to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, cystamine or cysteine analogs, wherein one or more hydrogen atoms are replaced deuterium or tritium, or one or more carbon atoms are replaced by carbon enriched at 13C or 14C are within the scope of this invention. These compounds are useful, for example, as analytical tools, probes in biological assays or compounds with an improved therapeutic profile.

It will be appreciated that the amount of cystamine analogues required for use in the treatment will vary not only. with the particular compound selected but also with the route of administration, the nature of the condition for which the treatment is required and the age and condition of the patient and will finally be at the discretion of the attending physician. In general, however, a suitable dose will be in the range of from about 0.1 to about 750 mg / kg of body weight per day, for example, in the range of 0.5 to 60 mg / kg / day or, for example, in the range from 1 to 20 mg / kg / day.

The desired dose can be presented conveniently in a single dose or as a divided dose which is administered at appropriate intervals, for example as two, three, four or more doses per day.

The cystamine analog is conveniently administered in a unit dosage form; for example containing from 5 to 2000 mg, from 10 to 1500 mg, conveniently from 20 to 1000 mg, much more conveniently from 50 to 700 mg of active ingredient per unit dosage form. In one embodiment, the cystamine analog is conveniently administered in a unit dosage form of 600 mg twice daily.

When the cystamine analogs or pharmaceutically acceptable salts thereof are used in combination with a second therapeutic agent that is active against Parkinson's disease, the dose of each compound can be either the same as or can differ from that when the compound It is used alone. The . Appropriate doses will be readily appreciated by those skilled in the field.

While it is possible that, for use in therapy, the cystamine analogs can be administered as the raw chemical, it is preferable to present the active ingredient as a pharmaceutical composition. In this way, the invention further provides a pharmaceutical composition comprising the cystamine analogs or a pharmaceutically acceptable salt of the present invention thereof together with one or more pharmaceutically acceptable carriers and therefore, optionally, other therapeutic and / or prophylactic ingredients. The carrier (s) must be "acceptable" in the sense that it is compatible with other ingredients of the formulation and is not harmful to the patient.

The pharmaceutical compositions include those which are suitable for oral, rectal, nasal, topical (including buccal and sublingual), transdermal, vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration or in a form suitable for administration by means of of inhalation or insufflation. Where appropriate, the compositions may be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the pharmacy field. All methods include the step of bringing the active ingredient into association with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired composition.

Pharmaceutical compositions that are suitable for oral administration may conveniently be presented as discrete units such as capsules, stamps or tablets each containing an amount predetermined active ingredient; as a powder or granules; as a solution, a suspension or as an emulsion. The active ingredient can also be presented as a bolus, electuary or paste. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants or wetting agents. The tablets may be coated according to methods well known in the art. The liquid oral preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs or may be presented as a dry product for constitution with water or other suitable vehicle before use. These liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils) or preservatives.

Cystamine analogs can also be formulated for parenteral administration (eg, by injection, eg bolus injection or continuous infusion) and can be presented as a unit dose form in ampoules, pre-filled syringes, infusion of small volume or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form, obtainable by aseptic isolation of a sterile solid or by lyophilization from a solution, for constitution with a suitable vehicle, eg, sterile free water. of pyrogens, before use.

For topical administration to the epidermis, the cystamine analogues can be formulated as ointments, creams or lotions, or as a transdermal patch. These transdermal patches may contain penetration stimulators such as linalool, carvacrol, thymol, citral, menthol and t-anethole. Ointments and creams can be formulated, for example, with an aqueous or oily base with the addition of suitable thickening and / or gelling agents. The lotions can be formulated with an aqueous or oily base and will also generally contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents and coloring agents.

Compositions which are suitable for topical administration in the mouth include rombotic pills comprising active ingredient in a flavored base, usually sucrose and gum arabic or tragacanth; pills comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and gum arabic; and mouth rinses comprising the active ingredient in a suitable liquid carrier.

Pharmaceutical compositions that are suitable for rectal administration in which the carrier is a solid are presented, for example, as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the field, and suppositories can be conveniently formed by mixing the active compound with the softened (s) or melted (s) carrier (s) by the cooling and the conformation in molds.

Compositions which are suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient these carriers which are known in the art to be appropriate.

For intranasal administration, the compounds or combinations can be used as a liquid atomization or a dispersible powder or in the form of drops. The droplets may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilizing agents or suspending agents. Liquid atomizations are conveniently supplied from packaging Pressurized For administration by inhalation, the compounds or combinations are conveniently supplied from an insufflator, nebulizer or pressurized pack or other conventional means to deliver an aerosol spray. The pressurized packages may comprise a propellant gas such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to supply a measured quantity.

Alternatively, for administration by means of inhalation or insufflation, the compounds or combinations may take the form of a dry powder composition, for example a mixture of powder of the compound and a suitable powder base such as lactose or starch. The powder composition can be presented in a unit dosage form in, for example, capsules or cartridges or for example gelatin or bubble-type packaging from which the powder can be administered with the aid of an inhaler or insufflator.

When desired, the compositions described above which are adapted to provide a sustained or modified release of the active ingredient. Examples of the cysteamine formulation are described, for example, in United States Publication 20090076166.

The present inventors have surprisingly discovered that cystamine has beneficial effects in parkinsonian animals when administered before and after the MPTP toxin capable of triggering the pathology. The present inventors have determined that cystamine can cancel out an initiated neurodegenerative process. As described in the examples, mice were intoxicated subcutaneously with MPTP following a 5-day regimen of 7 i.p. injections. of 20 mg / kg and administered 10 mg / kg of cystamine i.p. daily either 1) 2 days before the start of MPTP injections or 2) 24 hours after the last dose of MPTP and which continued for 14 days after the injury. At the end of the study, post mortem analyzes were performed to evaluate the state of the dopaminergic system (DAergic), more particularly centered on PARKINSON'S DISEASE. The present inventors surprisingly found that the i.p. of cystamine (10 mg / kg / day) to mice treated with MPTP starting after the deterioration of the nigrostriatal system (24 hours after treatment with MPTP), induced a significant recovery of the number of nigral dopaminergic neurons, evaluated by counting stereological of TH-immunoreactive cells, (p <0.05), of the number of cells expressing the DAT mRNA (p <0.05) as well as the Nurrl nigral mRNA levels (p <0.05). The present inventors have also determined that cystamine rescues dopaminergic neurons in the mouse model of unilateral striatal β-OHDA from Parkinson's disease. In addition, as shown in the examples herein, cystamine reverses the behavioral impairments that are induced by striatal 6-OHDA lesions in mice and restores some aspects of the dopaminergic nigral system and changes the striatal catecholaminergic contents and dynamics in the model. of unilateral striatal 6-OHDA mouse of Parkinson's disease. The present inventors have found that the role of the cystamine compounds is not limited solely to the preservation of existing neurons. The compounds can also reverse an induced apoptotic process and in this way can rescue damaged neurons from a degeneration that is being experienced.

Without wishing to be bound by any specific theory, the present inventors believe that cysteamine is the key neuroactive compound after the systemic administration of cystamine. Among the molecules investigated through HPLC measurements, which included cysteamine, cysteine, hypotaurine and taurine, it was discovered that cysteamine was the only one that increased significantly in the brains of natural mice after an i.p. single dose of 50 mg / kg and 200 mg / kg. In contrast, the levels of cysteine, hypotaurine and taurine remained unchanged or below the detection threshold. In addition, the present inventors have demonstrated that cysteamine crosses the BBB in a significant amount in vivo. These observations provide important information pertaining to the neuropharmacology of cysteamine and give additional support to its clinical relevance.

The BBB is a major obstacle to the clinical application of a large majority of potentially neuroactive compounds and when administered, it must be taken into account that the cystamine metabolite exerts its therapeutic effect. Amino acid transporters are well-studied components of BBB and comprise systems that prefer leucine, alanine, serine or cysteine (Wade and Katzman, 1975, Sershen and Lajtha 1979). It has been recognized that cysteine uses the system that prefers leucine to cross the BBB (Wade and Brady 1981). The ability of taurine to cross the BBB has also been described in rats using ISCP and apparently involves a system of affluence dependent on sodium and endothelial cell chlorine (Benrabh et al. 1995). On the other hand, the mechanism by which cysteamine can cross theBBB requires additional research. At this point, a quantitative and highly susceptible technique was used to study the transport through the BBB of cysteamine and cysteine. This was done without compromising the physical or functional integrity of the BBB and by circumventing peripheral metabolism processes associated with systemic administration. The inventors demonstrated the ability of cysteamine and cysteine to reach the brain in a significant amount.

The cysteamine uptake of the brain was further facilitated by the addition of cysteine in the perfusate.

In the above and in the following examples, all temperatures are exposed uncorrected in degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.

The following examples can be repeated with similar success by replacing the generically or specifically described reagents and / or the operating conditions of this invention with those used in the preceding examples.

EXAMPLES: EXAMPLE 1: Effect of Cystamine after Parkinsonism Induced by MPTP in Rodents Animals Young male C57BL / 6 mice (9 weeks of age, 25 grams) were purchased from Charles River Laboratories (Montréal, QC, Canada). The animals were housed 4 per cage under standard conditions with free access to food and water, were randomized and manipulated under the same conditions by a researcher. All the experiments were carried out in accordance with the Canadian Council on Animal Care and were approved by the Institutional Committee of the Center Hospitalier of I'Université Laval (CHUL, Québec, Canada). Throughout the experiment, the health status of all mice included in the study was closely monitored for weight loss or other symptoms of health-related problems. No effort was spared to minimize the pain and discomfort of the animals.

MPTP Administration Mice received 7 ip injections, two in the first 2 days of the experimental protocol in a 12-hour interval and once a day in 3 subsequent days, either 0.9% saline or MPTP-HC1 (20 mg / kg free base, Sigma, St. Louis, O) dissolved in freshly prepared 0.9% saline solution (Trernblay et al., 2006; Gibrat et al., 2007; Gibrat et al., 2010).

Treatments with cystamine The beneficial effects of cystamine in mice parkinsonians (cystamine dihydrochloride, Sigma, St. Louis, MO) were evaluated at a dose of 10 mg / kg dissolved in sterile 0.9% saline and freshly prepared for i.p. daily 1 hour before MPTP administration. The choice of dosage and the administration regimen were based on previous discoveries (Tremblay et al., 2006; Gibrat et al., 2010). The first injection of cystamine1 was administered either 1) 2 days before the start of MPTP injections (pre-treatment) or 2) 24 hours after the last dose of MPTP (post-treatment) and the treatment continued daily for 14 days. days after the injury.

This study was divided into 2 different experiments.

Experiment no.l. Neurorescape properties of the cystamine in mice injured with MPTP The effects of cystamine on the toxicity of MPTP were studied in the following experimental groups: Group I, Saline Solution + Saline Solution; Group II, Saline Solution + Cystamine after treatment; Group III; Saline solution + Cystamine pre-treatment; Group IV, MPTP + Saline Solution; Group V, MPTP + Cystamine after treatment, Group VI, MPTP + Cystamine pre-treatment. In total, 96 mice were used (n = 16 per group), monitored daily for weight variation and sacrificed finally by perfusion 24 hours after the last injection of cystamine (or vehicle).

Experiment no.2 Temporal course of the death of nigral energetic dopamine neurons induced by a subacute treatment with MPTP For this experiment, a total of 72 mice were used and divided into 6 groups (n = 12 per group). Groups I, II and III received subacute treatment with MPTP while Groups IV, V and VI were given 0.9% saline. Groups I and IV were sacrificed 24 hours, Groups II and V: 7 days were sacrificed and Groups III and VI: were sacrificed 14 days after the last injection of MPTP (or saline).

Perfusion and tissue processing The animals were sacrificed under deep anesthesia with ketamine / xylazine. (Vetalar, Bioniche, Belleville, ON / Rompun, Bayer, Toronto, ON) and perfused according to two methods under RNase-free conditions: Experiment 1. All mice were subjected to an intracardiac perfusion with saline buffered with RNase-free 0.1 M phosphate (PBS). After the intracardiac perfusion, the brains were collected and the two hemispheres separated. The left hemisphere was post-fixed in 4% paraformaldehyde (PFA) for 48 hours and transferred to 20% sucrose in 0.1 M PBS for cryoprotection. The coronal sections of the brain of 25 μp? of thickness were cut on a freezing microtome (Leica Microsystems, Montreal, QC) and serially collected in anti-freezing solution (0.2 M monobasic sodium monophosphate, 0.2 M dibasic sodium monophosphate, 30% ethylene glycol, 20% glycerol ) and kept at -20 ° C until use. The sections of the left hemisphere were used for additional immunohistochemistry and in situ hybridization protocols. The right hemispheres were instantly frozen in 2-methyl-butane and then stored at -80 ° C until dissected in a cryostat for HPLC and Western Blot (WB) analysis.

Experiment 2. In this experiment, the two hemispheres of each animal were instantly frozen and used for HPLC and WB analysis. The remaining 5 mice in each group were perfused intracardially by RNAse-free saline solution (0.9%) followed by 4% PFA, pH 7.4. After intracardiac perfusion, the brains were harvested and post-fixed in 4% PFA for 24 hours and transferred to 20% sucrose in 0.1 M PBS for cryoprotection. The brains were cut into coronal sections 25 μp \ thick. These sections were used for the immunohistochemistry and in situ hybridization required for the completion of experiment 2.

Quantification of catecholamine by means of HPLC The concentrations of striatal DA, acid 3,4- Dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) were measured by means of HPLC coupled with electrochemical detection (Calón et al., 2001; Calón et al., 2003). Each striatal sample comprised ten cryostat sections of 20 μ? T? of thickness of the structure that varies between the levels of +1.145 and +1.345 (Alien, 2008; Lein and collaborators, 2007). Two hundred μ? of perchloric acid (0.1 N; J.T. Baker) were added to each sample, which were homogenized and centrifuged (13000 xg) to generate a supernatant. Fifty μ? Supernatant of striatal tissues were injected directly into the chromatography system consisting of a Waters 717 plus automated automatic sampler injector, a Waters 1525 binary pump equipped with an Atlantis dC18 column (3 μ?), a Waters 2465 electrochemical detector and a vitreous carbon electrode (Waters Limited, Lachine, QC, Canada). The electrochemical potential was adjusted to 10 nA. The mobile phase consisted of 47.8 mM NaH2P04, 0.9 mM sodium octyl sulfate (J.T. Baker), 0.4 mM EDTA, 2 mM NaCl and 8% methanol (J.T. Baker) at pH 2.9 and was delivered at 1.0 ml / minute. The peaks were identified using the Breeze software (Waters). The HPLC quantifications were normalized to protein concentrations, determined with a protein assay kit with bicinchoninic acid (BCA) (Pierce, Rockford, IL, USA).

Immunohistochemistry of HT For the evaluation of the dopaminergic neuronal loss, the immunohistochemistry against the HT enzyme was carried out as previously described (Tremblay et al., 2006; Gibrat et al., 2009). In summary, sections that floated freely, after several washes and pre-incubation blocking, were incubated overnight at 4 ° C with a rabbit anti-TH antibody (Pel-Freez, Rogers, AR; 1: 5000). The sections were then incubated for 1 hour at room temperature (RT) in a solution containing biotinylated goat anti-mouse IgG (Vector Laboratories, Burlington, ON; 1: 1500) and subsequently placed in a solution containing contained avidin-biotin peroxidase complex (ABC Elite kit, Vector Laboratories, Burlington, ON) for 1 hour at room temperature. Finally, the reaction was developed in 3,3 '-diaminobenzidine tetrahydrochloride (DAB) solution (Sigma, St. Louis, O) and 0.1% 30% hydrogen peroxide (Sigma, St. Louis, MO) at temperature ambient. Other sections were treated as before except that the main antibody was omitted from the incubation medium. These sections remained virtually immune-free and served as negative controls. After the DAB reaction, the sections were mounted on gelatin-coated slides and counterstained with cresyl violet (Sigma, St. Louis, MO). All sections were finally air-dried, dehydrated in ascending degrees of ethanol, cleaned in xylene and placed on a coverslip with DPX mounting means (Electron icroscopy Science, Hatfield, PA).

In situ hybridization for Nurrl and DAT A specific probe of complementary RNA (cRNA) tagged with [35S] UTP was used to evaluate the levels of mRNA in the tissue of Nurrl, a nuclear receptor associated with the dopaminergic system (Zetterstrom et al., 1997). The cRNA probe for Nurrl arises from an EcoRI-BamHI fragment (accession number of gene bank: 1504-1907 NM_ 013613) of 403 bp of a full-length mouse Nurrl cDNA subcloned in pBluescript SK + and linearized with Xba I .

The DAT probe, a fragment of 2238 bp in length, was cloned into the pBluescript II SK + plasmid. The linearization was done with the Notl enzyme. An antisense probe was synthesized with [35 S] UTP and T7 RNA polymerase.

Perception probes were also generated for these markers and a specific signal was not obtained (data not shown). The brain sections were hybridized following the procedures described below and protocols previously published (Beaudry et al., 2000; Cossette et al., 2004; and collaborators, 2004).

This in situ protocol was conducted under RNAse free conditions. The cuts were mounted on Snowcoat X-tra ™ slides (Surgipath, Winnipeg, Canada) and stored under vacuum overnight before use. The brain sections were fixed in 4% PFA pH 7.4 at room temperature for 20 minutes. The pre-treatment was done with several consecutive baths (PBS 0.1 M twice for 5 minutes, proteinase K 0.1 g / ml 10 minutes at 37 ° C, acetylation bath (0.25% acetic anhydride, 0.1 M triethanolamine) 10 minutes, twice for 5 minutes in standard citrate saline (SSC) (0.3 M NaCl, 30 mM sodium citrate)). The successive baths of ethanol solutions (30%, 60%, 100%, 100%, 3 minutes each) were made for dehydration. In situ hybridization of the riboprobes in tissue sections was performed at 58 ° C overnight in a standard hybridization buffer (50% deionized formamide, 5 M sodium chloride, 1 M Tris, 0.5 M EDTA, 0.5 M solution), Denhart 50X, 50% dextran sulfate, 10 mg / mL tRNA, 1 M DTT, 35S coupled with 2 × 0.06 cpm / μ? Probe). Post-treatment was conducted using different successive baths: SSC 4X (30 minutes), removal of coverslips, 2X SSC twice (5 minutes), RNAse A 20 μg / mL (1 hour) at 37 ° C, milliQ water twice (15 seconds), SSC 2X (15 minutes), SSC 0.5X (30 minutes) at 60 ° C, SSC 0.1 X (30 minutes) at 60 ° C, SSC 0. IX (5 minutes) at room temperature. The repetitive baths of ethanol solutions (30%, 60%, 100%, 100%, 3 minutes each) were used for further dehydration. The tissue sections were then placed against radioactive sensitive films Biomax ™ (Kodak, New Haven, CT). The autoradiographies were developed using an exposure for 72 hours for Nurrl and an exposure for 5 hours for DAT.

The elimination of the fat was done with 4 ethanol baths, 2 xylene baths and 3 ethanol baths. After these steps, the slides were immersed in NTB emulsion (Kodak, New Haven, CT) fused at 42 ° C, air dried for 4 hours and stored in the dark for 5 days at 4 ° C. The emulsion was then developed (3.5 minutes) in developer D-19 (Kodak, New Haven, CT), rinsed in deionized water and fixed (5 minutes) in Kodak Rapid Fixer solution. The slides were rinsed in deionized water for 1 hour and then stained. The coloration was carried out using thionine (1 minute), followed by water and drops of ethanol then 3 ethanol baths (1 minute) and 3 xylene baths (3 minutes). The coverslips were attached to the slides with DPX mounting means.

Western Blot Analysis The samples were homogenized in 8 volumes of Lysis buffer (150 mM NaCl, 10 mM NaH2P04, 1% Triton X-100 (v / v), 0.5% SDS and 0.5% sodium deoxycholate) containing a cocktail of protease inhibitors (Roche, Mississauga, ON, Canada) and phosphatase inhibitors (Sigma, St-Louis, MO, USA). The samples were sonicated (3 x 10 seconds) and centrifuged at 100,000 g for 20 minutes at 4 ° C. The supernatant was collected and stored at -80 ° C. The protein concentration in each fraction was determined with a bicinchoninic acid protein assay kit. Twenty μg of total protein per sample were added to the Laemmli charge buffer and heated at 95 ° C for 5 minutes. The samples were then loaded and subjected to SDS-polyacrylamide gel electrophoresis (12%). Proteins were electrotransferred on Immobilon ™ 0.45 μm PVDF membranes (Millipore, Billerica, MA, USA) and blocked in 5% skimmed milk powder and 1% BSA in PBS IX for 1 hour. Membranes were immunoblotted with primary antibodies, rabbit anti-TH (Pel-Freez; 1: 5,000), rabbit anti-BAX (Cell signaling technology; Danvers, MA; 1: 1,000), rabbit anti-Bcl2 (Cell signaling technology; 1: 1,000), mouse anti-actin (ABM Inc., Richmond, BC, Canada; 1: 10,000) and with appropriate secondary antibodies, goat anti-rabbit or anti-mouse (Jackson Immunoresearch, West Grove, PA; 1: 100,000) followed by the addition of chemiluminescent reagents (KPL, Mandel • Scientific, Guelph, ON, Canada). The band intensities were quantified using an ImageQuant 4000 Digital Imaging System (Science Lab 2003 Image Gauge Software version 4.2, Fujifilm, New Haven, CT).

Densitometric measurements of Nurrl and DAT mRNA levels The levels of autoradiographic labeling were quantified by means of computerized densitometry. The digitized images of the brain and its analyzes were made with the same equipment as the one mentioned above. The optical density of the autoradiographs was translated into μ? / ^ Of-tissue using 14C radioactivity standards (ARC 146-14C standards, American Radiolabeled Chemicals Inc., St. Louis, MO). Nurrl and DAT mRNA levels were measured in the substantia nigra compacta (SNc) using similar antero-posterior levels for all sections. The average labeling for each level of SNc was calculated from 3 adjacent brain sections of the same mouse. Background intensities taken from white areas of the cross-linked substantia nigra (SNr) lacking the Nurrl or DAT mRNA levels were subtracted from each measurement.

Stereological quantification of reactive TH-immuno neurons The loss of dopaminergic neurons was determined by means of stereological counts of TH-immunoreactive cells (identifiable somas) under illumination of clear field. Each 10th section through the SNc was analyzed using the Stereo InvestigatorTM software (MicroBrightfield, Colchester, VT, USA) linked to an E800 NikonMR microscope (Nikon Canada Inc., Mississauga, ON, Canada). After delineation of SNc at low magnification (4X objective), a grid of points was placed on each section. For the most rostral level of the SNc analyzed (bregma -3.08 mm), the SNc was delineated by the visible limits with the medial terminal nucleus. For the intermediate levels (bregma -3.28 mm) and more flow of the SNc analyzed (bregma -3.58 mm), the structure was delimited by the exit of the third cranial nerve. The immunostained cells were counted by the optical dissector method at a higher magnification (20X objective). The counting variables were as follows: distance between counting frames (150 μ ?? X 150 um), size of counting frames (75 μ ??) and thickness of protection zone (1 μp). The cells were counted only if they did not intercept prohibited lines. The optical dissector method (Glaser and Glaser, 2000) was used to count cells positive for TH (positive for TH and cresyl violet) and negative for TH (only positive for cresyl violet). Stereological cell counts were performed stubbornly by two. independent researchers. It should be noted that the analyzes of the TH-immunoreactive profiles were restricted to the SNc and in this way the ventral tegmental area (VTA, for its acronym in English) was excluded.

Statistical analysis and image preparation All analyzes are expressed as means of groups ± S.E.M. The data belonging to the experiment no. 1 and no. 2 were evaluated by bidirectional ANOVA analysis. When the bidirectional ANOVA analysis produced non-significant interaction terms, the data were further analyzed for significance using Tukey's post hoc multiple comparison test. In all cases, a P value less than 0.05 was considered to be significant. Photomicrographs were taken using Picture Frame software (MicroBrightfield) linked to an E800 NikonMR microscope (Nikon Instruments, Toronto, ON). The images were finalized for illustration using Adobe Photoshop CS3.

RESULTS The effects of cystamine on the dopaminergic system Neuroprotective effects of cystamine in a subacute MPTP mouse model The histological evaluation of the endpoint was conducted in all the mice included in this study to investigate the beneficial effects of cystamine using several specific markers related to the DA system. TH is the speed limiting enzyme in the biosynthesis of DA and a marker for DA neurons. Nurrl is a transcription factor involved in the maintenance of the dopaminergic phenotype and the dopamine transporter, DAT, is a highly specific marker of pro-dopaminergic nigrostriatal neurons and in this way, its expression reflects the death state of dopaminergic neurons.

Treatment with MPTP generated a significant loss of TH-immunoreactive neurons that was associated with a concomitant loss of Nissl-stained neurons in the SNpc, consistent with a degeneration of DA neurons as opposed to a down regulation of TH expression ( p <0.001, Figure 1). This was accompanied by a significant decrease in the levels of Nurrl mRNA and nigral DAT in the SNpc (p <0.01, Figure 2, p <0.001, Figure 3). The daily drug administration of 10 mg / kg of cystamine started 2 days before the MPTP poisoning, confirming its neuroprotective action as revealed by the increase density of TH-immunoreactive cells in the SNpc (p <0.001, Figure 1 ), compared to animals not treated with MPTP. Post-mortem analysis of the DA system in mice pre-treated with cystamine further demonstrated the normalization of Nurrl mRNA levels (p <0.01, Figure 2), as well as the density of SNpc neurons expressing DAT (p <; 0.01, Figure 3).

Neuroresult potential of cystamine in a subacute MPTP mouse model The neurorescue properties of the cystamine treatment were evaluated starting 24 hours after the last MPTP injection. In mice post-treated with 10 mg / kg of cystamine, the dopaminergic neurotoxicity induced by MPTP was also significantly reduced. Mice treated with cystamine after MPTP injury exhibited a significantly higher number of neurons positive for TH and positive for Nissl (p <0.01, Figures 1A-1C) as well as a higher level of Nurrl mRNA (p <; 0.5, Figures 2A-2B) and DAT (p <0.5, Figures 3A-3C), comparable to those observed in mice not treated with MPTP.

Overall, evaluations of these three specific markers related to the DA system produced similar patterns and showed the beneficial effects of a post-MPTP treatment of cystamine, which do not confine cystamine to neuroprotection but rather extend the properties of cystamine to the neuro-rescue .

In order to conclude about the ability of cystamine to not only prevent (neuroprotection) but also to stop (neuro-rescue) the neurodegenerative process, the inventors conducted a study with the purpose of defining the time course of DA-related degeneration of the MPTP model used in these experiments.

Temporal course of the degeneration of nigral dopaminergic cells induced by the administration of subacute MPTP The loss of TH-positive and Nissl-positive neurons varied between 20% and 27% in the MPTP groups sacrificed from day 1 to day 14 after the last MPTP injection, but was only statistically significant on day 7 and 14 compared to the corresponding saline groups (p <0.01, Figures 4A-4C). In spite of the absence of a significant loss of TH-positive cells on day 1, a significant reduction in the levels of Nurrl and DAT mRNA in the SNpc was observed (p <0.05, Figures 5A-5B), which indicates some vulnerability of DA neurons. On the other hand, pro- and anti-apoptotic proteins, BAX and Bcl2, were increased and decreased respectively 24 hours after the last MPTP injection as evaluated by Western Blot analysis (p <0.05, Figures 6A -6C) . Taken together, these findings indicate that although dopaminergic neurons have not started to degenerate 24 hours after the last MPTP injection, they are involved in the apoptotic pathway. Notably, this supports that the beneficial effect of cystamine is of a nature of neurorescue RESULTS The effects of cystamine on the dopaminergic system EXAMPLE 2: Metabolism of cystamine and transport properties in the brain Animals and administration of cystamine Young male C57BL / 6 mice (9 weeks of age, 25 grams) were purchased from Charles River Laboratories (Montréal, QC, Canada). The animals were housed four per cage under standard conditions with free access to food and water, were randomized and manipulated under the same conditions by a researcher. All the experiments were carried out in accordance with the Canadian Council on Animal Care and were approved by the Institutional Committee of the Center Hospitalier of I'Université Laval (CHUL). Throughout the experiment, the health status of all mice included in the study was closely monitored. To clearly identify the active intermediate product after the injection of cystamine, as well as to understand its systemic and cerebral metabolism, an individual intraperitoneal (ip) injection of cystamine was administered to adult, normal C57BL / 6 male mice using three different doses: 10, 50 and 200 mg / kg, as determined by previous publications (Tremblay et al 2006, Gibrat et al 2010).

These doses were finally compared with injections of saline. The cystamine was dissolved in sterile saline (0.9%) and injected 1, 3, 12, 24 and 48 hours before annihilation. The animals were sacrificed under deep anesthesia with ketamine / xylazine and perfused through an intracardiac infusion with 0.1 M phosphate-buffered saline. After intracardiac perfusion, the brains were harvested, snap frozen in 2-methyl-butane and then they were stored at -80 ° C until the cryostat dissection for HPLC analysis. A total of 200 mice were assigned for this study (n = 10 per group).

Measurements of cysteine and cysteamine HFLC The HPLC associated with fluorescence detection was used in the quantification of cysteine and cysteamine from both sets of experiments: the dose response study and cerebral perfusion procedures in situ (ISCP, for its acronym in English) . The frontal cortex was homogenized in 200 μL of NaHCO 3 and then centrifuged at 15,700 g (4 ° C) for 20 minutes. Fifty μL of supernatant were directly derivatized with 30 μL of 4-fluoro-7-sulfamoylbenzo-furazane reagent (ABD-F). The alkylation reaction was completed at 55 ° C for 15 minutes and stopped with 4.9 μL of 12 N HCl. After centrifugation for 10 minutes at 7500 g (4 ° C), the supernatant was immediately injected into the chromatograph which It consisted of an automatic injector Waters 717 plus automatic sampler set at 4 ° C, a Waters 1525 binary pump equipped with an Atlantis dC18 column (3 μ ??; 3.9 x 150 mm) and a Waters 2487 Dual Absorbency detector (Waters limited, Lachine, QC, Canada) ). The excitation was adjusted to 385 nm and the emission to 515 nm. The mobile phase, which consisted of 2.5% methanol and 0.1 M ammonium acetate adjusted to pH 4.0, was supplied at 1 mL / minute (Santa et al. 2006). The peaks were identified and quantified using the Breeze ™ software (Waters limited). The HPLC quantifications were normalized to protein concentrations. Protein measurements were determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA) as described by the manufacturer's protocol.

HPLC measurements of taurine and hypothaurin Taurine and hypotaurine were measured by means of HPLC linked to UV detection. The supernatant of NaHCO3 brain extracts (bregma 1.54 to -0.58 mm) (see previous section for details) was directly derivatized with the reagent dansyl chloride (Sigma-Aldrich, St. Louis, MO, USA) based on methods published, modified (Sailer and Czupryna 1989, Calón and collaborators 1999). In summary, 50 L of dansyl chloride (1.2 mg / mL) and 50 μ ?, of sample or standard solution were mixed and then incubated for 30 minutes at 90 ° C. After the 7 centrifugation for 10 minutes at 7500 g (4 ° C), the supernatants were immediately injected into the chromatograph described above. The absorbance was adjusted to 337 nm and the sensitivity to a full scale absorbance unit 0.5. The mobile phase consisted of a water-acetonitrile mixture (88.5-11.35% v / v) containing 0.15% (v / v) phosphoric acid and was supplied at a rate of 0.8 mL / minute. The results were obtained using the same method described above.

Cerebral perfusion in situ Cerebral perfusion in situ (ISCP) was performed under deep anesthesia motivated by an i.p. of a mixture of ketamine / xylazine (140/8 mg / kg) and as previously described (Dagenais et al 2000; Ouellet et al. 2009). To ensure that 100% of the perfusate reached the BBB, a catheter was inserted into the carotid artery, common, right after ligature of the external branch (see Figure 3a for a schematic representation). Then the thorax was opened, the heart was removed and the perfusion started immediately at a flow rate of 2.5 mL / minute. The perfusion solution consisted of physiological saline buffered with bicarbonate: 128 mM NaCl, 24 mM NaH-C03, 4.2 mM KC1, 2.4 mM NaH2P04, 1.5 mM CaCl2, 0.9 mM MgCl2 and 9 mM D-glucose. The solution was gassed with 02 to 95% and C02 to 5% to obtain a pH of 7.4 and subsequently heated to 37 ° C. In all experiments, a radiolabeled tracer (14C-sucrose 0.3 μa / mL) was co-perfused with cysteamine (259 μ) and cysteine (165 μ), as a marker of BBB integrity and vascular volume. Four different groups of candid mice (n = 3) were evaluated in this study and perfused with cysteine, cysteamine, both molecules with 14C-sucrose alone which served as the control.

The procedure was terminated by decapitation of the mouse after 60 seconds of perfusion. The right cerebral hemisphere was collected and the frontal cortex was dissected and quickly frozen on dry ice for HPLC measurements of cysteine and cysteamine.

The remaining brain tissue of this hemisphere was digested in 2 mL of Solvable ™ (Perkin-Elmer Life Sciences, Waltham, MA, USA) at 50 ° C for 48 hours and mixed with 9 mL of Hisafe ™ scintillation cocktail (Perkin-Elmer Life Sciences). The aliquots of the perfusion fluid were taken before adding the radiolabelled marker for the HPLC quantification and after its passage through the syringe and catheter for the scintillation count, at the end of each experiment, for the calculation of the transport coefficient in the brain (see the following equation). The 14C isotope was counted in brain digestion and in the perfusate in a WallacMR scintillation counter (Perkin-Elmer Life Sciences). The clearance coefficients of cysteine and cysteamine uptake (Clup; μL / g / s) were calculated from the measured volume of distribution of cysteine and cysteamine, corrected with the vascular space determined with 14C-sucrose. The vascular space was constant and under 20 μL / g. The following equation was used for final calculations, as previously described (Dagenais et al. 2000).

Clup (μ? G'1 s'1) = Vd, in which Vd = cystein - Xi sucrose T Cpsrí. of cysteine Cperf. of sucrose Vd ^ L / g) represents the volume of distribution of the study compound, T (s) is the perfusion time, cysteine (ng / mg of tissue) or Sucrose (dpm / g) is the amount of cysteine or sucrose found in the frontal cortex or the remaining tissue of the hemisphere, respectively. C is the concentration (nq / μL; dpm / mL) in the perfusion solution (the cysteine serves as an example in the above equation).

Statistical data and analysis All data are expressed as group mean ± S.E.M. The data were evaluated by means of a bidirectional ANOVA analysis and analyzed by significance using the Student's t-test. Each group was compared with the 0 mg / kg group of associated time points (1, 3, 12, 24 or 48 hours). For the ISCP experiments, the Student t test was used to analyze the significance. In all the In cases, a p-value less than 0.05 was considered to be significant.

Results General effect of the administration of cystamine Throughout the dose response study, no deaths were reported and all mice exhibited good health except for the 200 mg / kg cystamine group where the mice exhibited symptoms of hypothermia (shuddering) and drowsiness (closing of the eyelids) ) during a period of approximately 2 hours, as previously reported (Gibrat et al. 2010).

Levels of cysteine and cysteamine in plasma and brain after administration of cystamine To investigate the metabolites found in the plasma and brain of injected mice. with a dose i.p. Individual cystamine, a highly susceptible HPLC method was used and allowed to specifically measure cysteine and cysteine through thiol derivatization (-SH) using an ABD-F compound before detection of fluorescence (Figure 7A). Cysteamine was not detectable in the plasma of mice treated with cystamine. Contrary to body expression, brain analyzes of cysteine and cysteine concentrations revealed a marked increase in cysteamine in the brain (Figure 7B). This increase was observed for the three doses of cystamine administered and at each time point set as the target by this dose response study. The bidirectional ANOVA analysis revealed significant differences for both factors; dose and time as well as a significant interaction between those two factors (p < 0.0001). Post hoc analyzes revealed significant increases specifically at 1 hour after injection for the doses of 50 mg / kg (p <0.05) and 200 mg / kg (p <0.01). Cysteamine levels remained significantly elevated 3 hours after the administration of cystamine (p <0.01) and progressively decreased over 48 hours, compared to the saline group. The administration of cystamine did not affect plasma cysteine levels (data not shown) or the brain, even at the highest dose of 200 mg / kg (Figure 7C). There were no indications of any significant change in plasma and / or cysteine levels for any of the doses or time points studied.

Levels of hipotaurine and taurine in plasma and brain after administration of cystamine Hypoturin is a major metabolite of cysteamine, which, in part, can generate taurine. To determine the concentration of these two molecules, a derivatization of primary amino groups with dansyl chloride was used before UV detection (Figure 8A).

This reaction takes place in both aromatic and aliphatic amine, which produces stable sulfonamide adducts and allows the detection of both hypotaurine (Figure 8A, compound 2) and taurine (Figure 8A, compound 1) within the same method. Despite some variation between the groups, no significant alteration of hipotaurine and taurine in the brain was observed for any of these three doses (10, 50 or 200 mg / kg) and in comparison with the control groups (Figs. 8B and 8C). There were no symptoms of hipotaurine and taurine accumulation in the brain at any of the time points of annihilation (1, 3, 12, 24 and 48 hours). Plasma levels remained below or close to the detection threshold.

Cysteine facilitates the transport of clsteamine in the brain Since a large majority of endogenous and exogenous compounds are inactive in the CNS because they do not cross the BBB, the inventors investigated the uptake of cysteine and cysteine in the brain. Using ISCP, the inventors measured the transport parameters of cysteine and cysteine in the blood-brain by directly infusing the brain via the carotid artery (Dagenais et al 2000; Ouellet et al. 2009) (Figure 9A). Both cysteine and cysteamine crossed the BBB, as observed by their transport coefficient in the brain (Clup), which corresponded to 4.39 ± 0.47 and 0.15 ± 0.02 μ ??, / ^ / d, respectively (Figures 9B and 9C). In comparison, a CNS drug routinely used, such as morphine, exhibits a Clup of 0.3 μm ^ _ ^ ^ / 3, while highly diffusible drugs such as diazepam or fatty acids exhibit a Clup which reaches up to 40 μ ?? / ^ /? (Bourasset et al 2003; Ouellet et al. 2009). Interestingly, the co-perfusion of cysteine and cysteamine increased the effect of its absorption in the brain. In fact, a significant Clup increase of cysteamine (+ 133%, p <0.05) and cysteine (+ 59% p <0.05) was measured when both compounds were injected simultaneously. Hypoturin and taurine, which have been shown to cross the BBB, were not reevaluated (Benrabh et al. 1995).

EXAMPLE 3 Properties of neurorescue and neuroreconstitution of cystamine after xntraestriatal administration of 6-OHDA Material and method Animals Young male C57BL / 6 mice (9 weeks of age, 25 grams) were purchased from Charles River Laboratories (Montréal, QC, Canada). The animals were housed 4 per cage under standard conditions with free access to food and water, were randomized and manipulated under the same conditions by a researcher. All the experiments were carried out in accordance with the Canadian Council on Animal Care and were approved by the Institutional Committee of the Center Hospitalier of I'Université Laval (CHUL). Throughout the experiment, the health status of all mice included in the study was closely monitored for weight loss or other symptoms of health-related problems. No effort was spared to minimize the pain and discomfort of the animals.

Treatment with 6-OHDA and cystamine This protocol was based on the intra-striatal, stereotactic, unilateral injection of 6-hydroxydopamine (6-OHDA). The intrastriatal administration of 6-OHDA creates degenerative, progressive and retrograde changes in the DA neurons of the substantia nigra (Bjorklund et al., 1997; Costantini et al., 2001). The mice were anesthetized using isoflurane and placed in a stereotactic frame adapted for mice. The 6-OHDA was dissolved at a concentration of 2 μg / μl in 0.9% saline and 0.02% ascorbic acid and injected with 2 μ? in the right striated body at a speed of 0.5 μ? / minute. The needle was left in this place for 3 minutes after injection before retraction. The injection was performed using a Hamilton syringe in accordance with the Stereotaxic coordinates: AP: +0.04 cm, ML: -0.18 cm, DV: -0.31 cm (corresponding to the Atlas of Alian P. 2008).

To serve as controls, other mice were subjected to the same surgical procedures, but only 2 μ? of solvent (0.9% saline solution and 0.02% ascorbic acid) in the same coordinates. The study was further divided into 2 different experiments: 1) The neurorescue effects of cystamine (cystamine dihydrochloride, Sigma, St. Louis, MO) in mice injured with 6-OHDA. For this experiment, the first injection i.p. of 10 mg / kg of cystamine was administered 3 days after surgery and the treatment continued daily for 14 days. 2) The effects of neuro-restoration of cystamine (cystamine dihydrochloride, Sigma, St. Louis, MO) in mice injured with 6-OHDA. The treatment of cystamine began 3 weeks after surgery, when it is known that the dopaminergic lesion (DAergic) is stable and has reached its peak. The treatment was continued daily for 6 weeks.

The effects of cystamine on the toxicity of 6-OHDA were studied in the following experimental groups: Group I, Drill + Saline Solution; Group II, 6-OHDA + Saline Solution; Group III; Drill + Cystamine; Group IV, 6-OHDA + Cystamine In total, 40 mice (n = 10 per group) were used for each experiment, monitored daily for weight variation and finally sacrificed by perfusion 24 hours after the last injection of cystamine (or vehicle). The results are shown in the Figures 10A-10E, 11A-11D and 12A-12.

Perfusion and tissue processing The animals were sacrificed under deep anesthesia with ketamine / xylazine (Vetalar, Bioniche, Belleville, ON / Rompun, Bayer, Toronto, ON). All mice were subjected to an intracardiac perfusion with saline buffered 0.1 phosphate RNase free (PBS). After intracardiac perfusion, the brains were harvested and the hindbrain of each mouse, which comprised the entire mesencephalon, was post-fixed in 4% PFA for further analysis of immunohistochemistry and in situ hybridization. The anterior brain, which comprises the right and left striatum, was instantly frozen and used for HPLC and WB analysis.

The quantification of catecholamine by means of HPLC, the immunohistochemistry of TH and the in situ hybridization for Nurrl and DAT were conducted as described in Example 1.

Densitometric measurements of levels of Nurrl and DAT mRNA The levels of autoradiographic labeling were quantified by means of computerized densitometry.

The digitized images of the brain and its analysis were made with the same equipment as the one mentioned above. The optical density of the autoradiographs was translated into μ ?? / ^ of tissue using 14C radioactivity standards (ARC 146-14C standards, American Radiolabeled Chemicals Inc., St. Louis, MO). Nurrl and DAT mRNA levels were measured in the substantia nigra compacta (SNc) using similar antero-posterior levels for all sections. The average labeling for each level of SNc was calculated from 3 adjacent brain sections of the same mouse. Background intensities taken from white areas of the substantia nigra reticulata (SNr) lacking the Nurrl or DAT mRNA levels were subtracted from each measurement.

Stereological quantification of TH-immunoreactive neurons The loss of dopaminergic neurons was determined by means of stereological counts of TH-immunoreactive cells (identifiable somas) under brightfield illumination. Each 10th section through the SNc was analyzed using the Stereo InvestigatorTM software (MicroBrightfield, Colchester, VT, USA) linked to an E800 NikonMR microscope (Nikon Canada Inc., Mississauga, ON, Canada). After delineation of SNc at low magnification (4X objective), a grid of points was placed on each section. For the most rostral level of SNc analyzed (bregma -3.08 mm), the SNc was delineated by the visible limits with the medial terminal nucleus. For the intermediate levels (bregma -3.28 mm) and more flow of the SNc analyzed (bregma -3.58 mm), the structure was delimited by the exit of the third cranial nerve. The immunostained cells were counted by the optical dissector method at a higher magnification (20X objective). The counting variables were as follows: distance between counting frames (150 μp? X 150 μp?), Size of counting frames (75 um) and thickness of protection zone (1 μp?). The cells were counted only if they did not intercept prohibited lines. The optical dissector method (Glaser and Glaser, 2000) was used to count the TH-positive and cresyl violet-positive and TH-negative expression cells (only positive for cresyl violet). Stereological cell counts were performed stubbornly by two independent investigators. It should be noted that the analyzes of the TH-immunoreactive profiles were restricted to the SNc and thus the area of the ventral tegmentum (VTA) was excluded.

Rotation induced by apomorphine The rotational behavior is considered to represent a reliable physiological measure of DA depletion and asymmetric stimulation of DA receptors. When applying DA agonists such as apomorphine (Anden and collaborators, 1966; Ungerstedt et al., 1968), the DA receptors are stimulated directly which leads to a rotation contralateral to the hemisphere depleted in DA. The mice were challenged with apomorphine (0.5 mg / kg, Sigma-Aldrich) at 3, 6 and 9 weeks after the injury and the rotating behaviors were evaluated for 45 minutes using an automated rotometer system. The results were averaged and expressed as a net rotation (= number of contralateral rotations - number of homolateral rotations) as previously described (Metz 2002). The animals were allowed to become habituated for 5 minutes after the injection before the recording of rotations began.

Proof of displacement The step adjustment test was adapted from studies in rats (Lindner et al., 1995) and mice treated with MPTP (Blume et al., 2009). The mice were held at the base of the tail with their hind legs suspended on the table and moved backward at a constant speed so that they traveled 1 meter away for 3-4 s. The mice were recorded on video using a digital video camera (Sony Handcam, DCR-HC90E PAL), but the video was analyzed offline by counting the step adjustment number made with the contralateral or ipsilateral foot, in relation to the hemisphere injured, about the total distance. Three trials were carried out in mice at 3 weeks, 6 weeks and 9 weeks after surgery and the intermediate data were calculated in all the trials.

Cylinder test The limb use asymmetry test (cylinder), which measures the movements of the forelimbs that carry the weight during erecting, has been shown to be a good indicator of the loss of nigrostriatal cells in rats and mice injected with 6 - Unilateral OHDA (Schallert et al., 2000, Tillerson et al., 2001, Lundblad et al., 2004, Lancu et al., 2005). To examine the lateral deviation in the spontaneous use of the forelimbs during the exploration activity, the mice were placed individually inside a glass cylinder (10 centimeters in diameter, 14 centimeters in height), which was located in front of two vertical mirrors in order to observe mice from all angles. The mice were videotaped immediately for 3 minutes. The habituation of the animals to the test cylinder was not allowed before the video recording. The video recordings were then examined to count the number of touches on the support wall (contacts with fully extended fingers) executed independently with the ipsilateral and contralateral forelimb with respect to the lesion. A measure of the asymmetry of the use of the forelimbs was obtained by expressing the touches made by the contralateral leg with respect to the lesion (right leg) as a percentage of the total number of touches in each lesion.

Statistical analysis All analyzes are expressed as group mean ± S.E. . The data was evaluated by means of a bidirectional ANOVA analysis. When the bidirectional ANOVA analysis produced non-significant interaction terms, the data were further analyzed for significance using Tukey's post hoc multiple comparison test. In all cases, a P value less than 0.05 was considered to be significant.

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It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (32)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. The use of a therapeutically effective amount of at least one cystamine analog or a pharmaceutically acceptable salt thereof, to modify the progress of Parkinson's disease in a patient.

2. The use of a therapeutically effective amount of at least one cystamine analog or a pharmaceutically acceptable salt thereof as a neurorescue agent to modify the progress of Parkinson's disease in a patient.

3. The use of a therapeutically effective amount of at least one cystamine analog or a pharmaceutically acceptable salt thereof as a neurorestoration agent to modify the progress of Parkinson's disease in a patient.

4. The use according to any of claims 1 to 3, to reduce fatigue in a patient with Parkinson's disease.

5. The use according to any of claims 1 to 3, to reduce the severity of non-motor symptoms in a patient with Parkinson's disease.

6. The use according to any of claims 1 to 3, to reduce functional decay in a patient with Parkinson's disease.

7. The use according to any of claims 1 to 3, to reduce the clinical progress of the disease.

8. The use according to any of claims 1 to 3, to slow down the clinical progress of the disease.

9. The use according to any of claims 1 to 8, wherein the patient is a patient with Parkinson's disease in early stage.

10. The use according to any of claims 1 to 8, wherein the patient with Parkinson's disease in the initial stage is a stage III or IV patient according to the classification of Hoehn and Yahr.

11. The use according to any of claims 1 to 8, wherein the patient with Parkinson's disease is a patient with stage III according to the classification of Hoehn and Yahr.

12. The use according to any of claims 1 to 11, wherein the therapeutically effective amount of at least one cystamine analog or a pharmaceutically acceptable salt thereof. it is in the range of about 0.1 to about 750 mg / kg of body weight per day.

13. The use according to any of claims 1 to 11, wherein the therapeutically effective amount of at least one cystamine analogue or a pharmaceutically acceptable salt thereof is in the range of about 0.5 to about 60 mg / kg / day.

14. The use according to any of claims 1 to 11, wherein the therapeutically effective amount of at least one cystamine analogue or a pharmaceutically acceptable salt thereof is in the range of about 1 to about 20 mg / kg / day.

15. The use according to any of claims 1 to 11, wherein the therapeutically effective amount of at least one cystamine analog or a pharmaceutically acceptable salt thereof is suitable to be administered in a unit dosage form containing 5 to 2000 mg of active ingredient per unit dosage form.

16. The use according to any of claims 1 to 11, wherein the therapeutically effective amount of at least one cystamine analog or a pharmaceutically acceptable salt thereof is suitable to be administered in a unit dosage form containing 10 to 1500 mg of active ingredient per unit dosage form.

17. The use according to any of claims 1 to 11, wherein the therapeutically effective amount of at least one cystamine analog or a pharmaceutically acceptable salt thereof is suitable to be administered in a unit dosage form containing 20 to 1000 mg of active ingredient per unit dosage form.

18. The use according to any of claims 1 to 11, wherein the therapeutically effective amount of at least one cystamine analog or a pharmaceutically acceptable salt thereof is suitable to be administered in a unit dosage form containing 50 to 700 mg of active ingredient per unit dosage form.

19. A combination, characterized in that it comprises at least one cystamine and cysteine analog or pharmaceutically acceptable salts thereof to modify the progress of Parkinson's disease.

20. The combination according to claim 19, characterized in that the cystamine and cysteine analog are present in a ratio of 10: 1 to 1:10 of cysteine and cysteine analog, respectively.

21. The combination according to claim 19, characterized in that the cystamine and cysteine analog are present in a ratio of 1: 1.

22. The combination according to claims 19, 20 or 21, characterized in that the cystamine analog and the additional agent are suitable for sequential use.

23. The combination according to claims 19, 20 or 21, characterized in that the cystamine analog and the additional agent are suitable for simultaneous use.

24. A pharmaceutical composition, characterized in that it comprises at least one cystamine analog or pharmaceutically acceptable salts thereof and comprising cysteine or a pharmaceutically acceptable salt thereof.

25. The composition according to claim 24, characterized in that the cystamine and cysteine analog are present in a ratio of 10: 1 to 1:10 of the cystamine and cysteine analog, respectively.

26. The composition according to claim 24, characterized in that the cystamine and cysteine analog are present in a ratio of 1: 1.

27. The composition according to claims 24, 25 or 26, characterized in that the cystamine analog and the additional agent are suitable for used sequentially

28. The composition according to claims 24, 25 or 26, characterized in that the cystamine analog and the additional agent are suitable for simultaneous use.

29. The use according to any one of claims 1 to 18, the composition according to any of claims 24 to 28 or the combination according to any of claims 19 to 23, wherein the cystamine analogue is cysteamine, cystamine, taurine or hypothaurin or a pharmaceutically acceptable salt thereof.

30. The use according to any one of claims 1 to 18, the composition according to any of claims 24 to 28 or the combination according to any of claims 19 to 23, wherein the cystamine analog is cystamine or cysteamine or a pharmaceutically acceptable salt thereof.

31. The use according to any of claims 1 to 18, the composition according to any of claims 24 to 28 or the combination according to any of claims 19 to 23, wherein the cystamine analogue is cystamine or a salt pharmaceutically acceptable thereof.

32. The use according to any one of claims 1 to 18, the composition according to any of claims 24 to 28 or the combination according to any of claims 19 to 23, wherein the cystamine analogue is cysteamine or a salt pharmaceutically acceptable thereof.